UCSF RNA Journal Club

A newsletter announcing the next presenter for RNA Journal Club

Malin Akerblom

Loss of a mammalian circular RNA locus causes miRNA deregulation and affects brain function
Piwecka M1, Glažar P1, Hernandez-Miranda LR2, Memczak S1,3, Wolf SA4, Rybak-Wolf A1, Filipchyk A1, Klironomos F1, Cerda Jara CA1, Fenske P5, Trimbuch T5, Zywitza V1, Plass M1, Schreyer L1, Ayoub S1, Kocks C1, Kühn R6,7, Rosenmund C5, Birchmeier C2, Rajewsky N8.
Science. 2017 Aug 10. pii: eaam8526. doi: 10.1126/science.aam8526. [Epub ahead of print]
August 10, 2017
Laboratory for Systems Biology of Gene Regulatory Elements, Berlin Institute for Medical Systems Biology, Max Delbrück Center for Molecular Medicine, Robert-Rössle-Str. 10, Berlin-Buch, Germany. Laboratory for Developmental Biology and Signal Transduction, Max Delbrück Center for Molecular Medicine, Robert-Rössle-Str. 10, Berlin-Buch, Germany. Experimental and Clinical Research Center, a joint cooperation between the Charité Medical Faculty and the Max Delbrück Center for Molecular Medicine, Berlin, Germany. Laboratory for Cellular Neurosciences, Max Delbrück Center for Molecular Medicine, Robert-Rössle-Str. 10, Berlin-Buch, Germany. Department of Neurophysiology, NeuroCure Cluster of Excellence, Charité-Universitätsmedizin, Berlin, Germany. Transgenic Core Facility, Max Delbrück Center for Molecular Medicine, Robert-Rössle-Str. 10, Berlin-Buch, Germany. Berlin Institute of Health, Kapelle-Ufer 2, Berlin, Germany. Laboratory for Systems Biology of Gene Regulatory Elements, Berlin Institute for Medical Systems Biology, Max Delbrück Center for Molecular Medicine, Robert-Rössle-Str. 10, Berlin-Buch, Germany. [email protected]
Hundreds of circular RNAs (circRNAs) are highly abundant in mammalian brain, with oftentimes conserved expression. Here, we show that the circRNA Cdr1as is massively bound by miR-7 and miR-671 in the human and mouse brain. When the Cdr1as locus was removed from the mouse genome, knockout animals displayed impaired sensorimotor gating, a deficit in the ability to filter out unnecessary information associated with neuropsychiatric disorders. Electrophysiological recordings revealed dysfunctional synaptic transmission. Expression of microRNAs miR-7 and miR-671 was specifically and post-transcriptionally misregulated in all brain regions analyzed. Expression of immediate early genes such as Fos, a direct miR-7 target, was enhanced in Cdr1as-deficient brains, providing a possible molecular link to the behavioral phenotype. Our data indicate an in vivo loss-of-function circRNA phenotype and suggest that interactions between circRNAs and miRNAs are important for normal brain function.
Date: 
August 23, 2017
Where: 
HSW 1057 at noon

Bin Zhang

Structural basis of CRISPR–SpyCas9 inhibition by an anti-CRISPR protein
Dong1, Guo M1, Wang S1, Zhu Y1, Wang S1, Xiong Z1, Yang J1, Xu Z1, Huang Z1.
Nature. 2017 Jun 15;546(7658):436-439. doi: 10.1038/nature22377. Epub 2017 Apr 27.
June 15, 2017
HIT Center for Life Sciences, School of Life Science and Technology, Harbin Institute of Technology, Harbin 150080, China.
CRISPR-Cas9 systems are bacterial adaptive immune systems that defend against infection by phages. Through the RNA-guided endonuclease activity of Cas9 they degrade double-stranded DNA with a protospacer adjacent motif (PAM) and sequences complementary to the guide RNA. Recently, two anti-CRISPR proteins (AcrIIA2 and AcrIIA4 from Listeria monocytogenes prophages) were identified, both of which inhibit Streptococcus pyogenes Cas9 (SpyCas9) and L. monocytogenes Cas9 activity in bacteria and human cells. However, the mechanism of AcrIIA2- or AcrIIA4-mediated Cas9 inhibition remains unknown. Here we report a crystal structure of SpyCas9 in complex with a single-guide RNA (sgRNA) and AcrIIA4. Our data show that AcrIIA2 and AcrIIA4 interact with SpyCas9 in a sgRNA-dependent manner. The structure reveals that AcrIIA4 inhibits SpyCas9 activity by structurally mimicking the PAM to occupy the PAM-interacting site in the PAM-interacting domain, thereby blocking recognition of double-stranded DNA substrates by SpyCas9. AcrIIA4 further inhibits the endonuclease activity of SpyCas9 by shielding its RuvC active site. Structural comparison reveals that formation of the AcrIIA4-binding site of SpyCas9 is induced by sgRNA binding. Our study reveals the mechanism of SpyCas9 inhibition by AcrIIA4, providing a structural basis for developing 'off-switch' tools for SpyCas9 to avoid unwanted genome edits within cells and tissues.
Date: 
August 16, 2017
Where: 
HSW 1057 at noon

Vanille Greiner

Correction of a pathogenic gene mutation in human embryos
Hong Ma1*, Nuria Marti-Gutierrez1*, Sang-Wook Park2*, Jun Wu3*, Yeonmi Lee1, Keiichiro Suzuki3, Amy Koski1, Dongmei Ji1, Tomonari Hayama1, Riffat Ahmed1, Hayley Darby1, Crystal Van Dyken1, Ying Li1, Eunju Kang1, A.-Reum Park2, Daesik Kim4, Sang-Tae Kim2, Jianhui Gong5,6,7,8, Ying Gu5,6,7, Xun Xu5,6,7, David Battaglia1,9, Sacha A. Krieg9, David M. Lee9, Diana H. Wu9, Don P. Wolf1, Stephen B. Heitner10, Juan Carlos Izpisua Belmonte3§, Paula Amato1,9§, Jin-Soo Kim2,4§, Sanjiv Kaul10§ & Shoukhrat Mitalipov1,10§
Nature. Received: March 28, 2017. Accepted June 27, 2017. Published online August 2, 2017.
August 2, 2017
1Center for Embryonic Cell and Gene Therapy, Oregon Health & Science University, 3303 Southwest, Bond Avenue, Portland, Oregon 97239, USA. 2Center for Genome Engineering, Institute for Basic Science, 70, Yuseong-daero 1689-gil, Yuseong-gu, Daejeon, 34047, Republic of Korea. 3Gene Expression Laboratory, Salk Institute for Biological Studies, 10010 North Torrey Pines Road, La Jolla, California 92037, USA. 4Department of Chemistry, Seoul National University, 599 Gwanak-ro, Gwanak-gu, Seoul, 151-747, Republic of Korea. 5BGI-Shenzhen, Build 11, Beishan Industrial Zone, Yantian District, Shenzhen, 518083, China. 6China National GeneBank, BGI-Shenzhen, Jinsha Road, Dapeng District, Shenzhen, 518210, China. 7BGI-Qingdao, 2877 Tuanjie Road, Sino- German Ecopark, Qingdao, 266000, China. 8Shenzhen Engineering Laboratory for Innovative Molecular Diagnostics, BGI-Shenzhen, Build 11, Beishan Industrial Zone, Yantian District, Shenzhen, 518083, China. 9Division of Reproductive Endocrinology and Infertility, Department of Obstetrics and Gynecology, Oregon Health & Science University, 3303 Southwest, Bond Avenue, Portland, Oregon 97239, USA. 10Knight Cardiovascular Institute, Oregon Health & Science University, 3181 Southwest, Sam Jackson Park Road, Portland, Oregon 97239, USA. * These authors contributed equally to this work. §These authors jointly supervised this work.
Genome editing has potential for the targeted correction of germline mutations. Here we describe the correction of the heterozygous MYBPC3 mutation in human preimplantation embryos with precise CRISPR–Cas9-based targeting accuracy and high homology-directed repair efficiency by activating an endogenous, germline-specific DNA repair response. Induced double-strand breaks (DSBs) at the mutant paternal allele were predominantly repaired using the homologous wild-type maternal gene instead of a synthetic DNA template. By modulating the cell cycle stage at which the DSB was induced, we were able to avoid mosaicism in cleaving embryos and achieve a high yield of homozygous embryos carrying the wild-type MYBPC3 gene without evidence of off-target mutations. The efficiency, accuracy and safety of the approach presented suggest that it has potential to be used for the correction of heritable mutations in human embryos by complementing preimplantation genetic diagnosis. However, much remains to be considered before clinical applications, including the reproducibility of the technique with other heterozygous mutations.
Date: 
August 9, 2017
Where: 
HSW 1057 at noon

Roman Camarda

Exosome RNA Unshielding Couples Stromal Activation to Pattern Recognition Receptor Signaling in Cancer
Barzin Y. Nabet, Yu Qiu, Jacob E. Shabason, Tony J. Wu, Taewon Yoon, Brian C. Kim, Joseph L. Benci, Angela M. DeMichele, Julia Tchou, Joseph Marcotrigiano, and Andy J. Minn
Cell
July 27, 2017
Department of Radiation Oncology Department of Medicine Department of Surgery Institute for Immunology Parker Institute for Cancer Immunotherapy Basser Center for BRCA Abramson Cancer Center Abramson Family Cancer Research Institute Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA Department of Chemistry and Chemical Biology, Rutgers University, Piscataway, NJ, USA
Interactions between stromal fibroblasts and cancer cells generate signals for cancer progression, therapy resistance, and inflammatory responses. Although endogenous RNAs acting as damage-associated molecular patterns (DAMPs) for pattern recognition receptors (PRRs) may represent one such signal, these RNAs must remain unrecognized under non-pathological conditions. We show that triggering of stromal NOTCH-MYC by breast cancer cells results in a POL3-driven increase in RN7SL1, an endogenous RNA normally shielded by RNA binding proteins SRP9/14. This increase in RN7SL1 alters its stoichiometry with SRP9/14 and generates unshielded RN7SL1 in stromal exosomes. After exosome transfer to immune cells, unshielded RN7SL1 drives an inflammatory response. Upon transfer to breast cancer cells, unshielded RN7SL1 activates the PRR RIG-I to enhance tumor growth, metastasis, and therapy resistance. Corroborated by evidence from patient tumors and blood, these results demonstrate that regulation of RNA unshielding couples stromal activation with deployment of RNA DAMPs that promote aggressive features of cancer.
Date: 
August 2, 2017
Where: 
HSW 1057 at noon

Ryan Wagner

TBA
Date: 
July 26, 2017
Where: 
HSW 1057 at noon

Gabriel Eades

TERRA RNA Antagonizes ATRX and Protects Telomeres
Hsueh-Ping Chu, Catherine Cifuentes-Rojas, Barry Kesner, Eric Aeby, Hun-goo Lee, Chunyao Wei, Hyun Jung Oh, Myriam Boukhali, Wilhelm Haas, Jeannie T. Lee
Cell
June 29, 2017
1Howard Hughes Medical Institute, Boston, MA 02114, USA 2Department of Molecular Biology, Massachusetts General Hospital, Boston, MA 02114, USA 3Department of Genetics, Harvard Medical School, Boston, MA 02114, USA 4Massachusetts General Hospital Cancer Center, Charlestown, Boston, MA 02114, USA 5Department of Medicine, Harvard Medical School, Boston, MA 02114, USA
Through an integration of genomic and proteomic approaches to advance understanding of long noncoding RNAs, we investigate the function of the telomeric transcript, TERRA. By identifying thousands of TERRA target sites in the mouse genome, we demonstrate that TERRA can bind both in cis to telomeres and in trans to genic targets. We then define a large network of interacting proteins, including epigenetic factors, telomeric proteins, and the RNA helicase, ATRX. TERRA and ATRX share hundreds of target genes and are functionally antagonistic at these loci: whereas TERRA activates, ATRX represses gene expression. At telomeres, TERRA competes with telomeric DNA for ATRX binding, suppresses ATRX localization, and ensures telomeric stability. Depleting TERRA increases telomerase activity and induces telomeric pathologies, including formation of telomere-induced DNA damage foci and loss or duplication of telomeric sequences. We conclude that TERRA functions as an epigenomic modulator in trans and as an essential regulator of telomeres in cis.
Date: 
July 19, 2017
Where: 
HSW 1057 at noon

Hui Li

Exosomes facilitate therapeutic targeting of oncogenic KRAS in pancreatic cancer
Kamerkar S, LeBleu VS, Sugimoto H, Yang S, Ruivo CF, Melo SA, Lee JJ, Kalluri R.
Nature. 2017 Jun 22;546(7659):498-503. doi: 10.1038/nature22341. Epub 2017 Jun 7.
Department of Cancer Biology, Metastasis Research Center, University of Texas MD Anderson Cancer Center, Houston, Texas 77005, USA. Instituto de Investigação e Inovação em Saúde, Universidade do Porto, Portugal (I3S), 4200 Porto, Portugal; Institute of Pathology and Molecular Immunology of the University of Porto (IPATIMUP), 4200 Porto, Portugal. Department of Biostatistics, University of Texas MD Anderson Cancer Center, Houston, Texas 77005, USA.
The mutant form of the GTPase KRAS is a key driver of pancreatic cancer but remains a challenging therapeutic target. Exosomes are extracellular vesicles generated by all cells, and are naturally present in the blood. Here we show that enhanced retention of exosomes, compared to liposomes, in the circulation of mice is likely due to CD47-mediated protection of exosomes from phagocytosis by monocytes and macrophages. Exosomes derived from normal fibroblast-like mesenchymal cells were engineered to carry short interfering RNA or short hairpin RNA specific to oncogenic KrasG12D, a common mutation in pancreatic cancer. Compared to liposomes, the engineered exosomes (known as iExosomes) target oncogenic KRAS with an enhanced efficacy that is dependent on CD47, and is facilitated by macropinocytosis. Treatment with iExosomes suppressed cancer in multiple mouse models of pancreatic cancer and significantly increased overall survival. Our results demonstrate an approach for direct and specific targeting of oncogenic KRAS in tumours using iExosomes.
Date: 
July 12, 2017
Where: 
HSW 1057 at noon

Fang Huang

Extensive RNA editing and splicing increase immune self-representation diversity in medullary thymic epithelial cells
Danan-Gotthold M, Guyon C, Giraud M, Levanon EY, Abramson J.
Genome Biol. 2016 Oct 24;17(1):219.
The Mina and Everard Goodman Faculty of Life Sciences, Bar-Ilan University, Ramat-Gan, 52900, Israel. Department of Infection Immunity and Inflammation, Cochin Institute, Paris, France. The Mina and Everard Goodman Faculty of Life Sciences, Bar-Ilan University, Ramat-Gan, 52900, Israel. [email protected] Department of Immunology, Weizmann Institute of Science, Rehovot, Israel. [email protected]
BACKGROUND: In order to become functionally competent but harmless mediators of the immune system, T cells undergo a strict educational program in the thymus, where they learn to discriminate between self and non-self. This educational program is, to a large extent, mediated by medullary thymic epithelial cells that have a unique capacity to express, and subsequently present, a large fraction of body antigens. While the scope of promiscuously expressed genes by medullary thymic epithelial cells is well-established, relatively little is known about the expression of variants that are generated by co-transcriptional and post-transcriptional processes. RESULTS: Our study reveals that in comparison to other cell types, medullary thymic epithelial cells display significantly higher levels of alternative splicing, as well as A-to-I and C-to-U RNA editing, which thereby further expand the diversity of their self-antigen repertoire. Interestingly, Aire, the key mediator of promiscuous gene expression in these cells, plays a limited role in the regulation of these transcriptional processes. CONCLUSIONS: Our results highlight RNA processing as another layer by which the immune system assures a comprehensive self-representation in the thymus which is required for the establishment of self-tolerance and prevention of autoimmunity. KEYWORDS: Alternative splicing; Medullary thymic epithelial cells (mTECs); RNA editing; RNA sequencing; Self-tolerance; Thymus
Date: 
July 5, 2017
Where: 
HSW 1057 at noon

Theodore Roth

Mapping the genomic landscape of CRISPR–Cas9 cleavage
Peter Cameron, Chris K Fuller, Paul D Donohoue, Brittnee N Jones, Matthew S Thompson, Matthew M Carter, Scott Gradia, Bastien Vidal, Elizabeth Garner, Euan M Slorach, Elaine Lau, Lynda M Banh, Alexandra M Lied, Leslie S Edwards, Alexander H Settle, Daniel Capurso, Victor Llaca, Stéphane Deschamps, Mark Cigan, Joshua K Young & Andrew P May.
Nature Methods
June 1, 2017
Caribou Biosciences, Berkeley, California, USA. DuPont Pioneer, Johnston, Iowa, USA. Present addresses: Omicia, Inc., Oakland, California, USA (B.N.J.); Genus Research, DeForest, Wisconsin, USA (M.C.) and Chan Zuckerberg Biohub, San Francisco, California, USA (A.P.M.). These authors contributed equally to this work. Correspondence should be addressed to J.K.Y. ([email protected]) or A.P.M. ([email protected]).
RNA-guided CRISPR–Cas9 endonucleases are widely used for genome engineering, but our understanding of Cas9 specificity remains incomplete. Here, we developed a biochemical method (SITE-Seq), using Cas9 programmed with single-guide RNAs (sgRNAs), to identify the sequence of cut sites within genomic DNA. Cells edited with the same Cas9–sgRNA complexes are then assayed for mutations at each cut site using amplicon sequencing. We used SITE-Seq to examine Cas9 specificity with sgRNAs targeting the human genome. The number of sites identified depended on sgRNA sequence and nuclease concentration. Sites identified at lower concentrations showed a higher propensity for off-target mutations in cells. The list of off-target sites showing activity in cells was influenced by sgRNP delivery, cell type and duration of exposure to the nuclease. Collectively, our results underscore the utility of combining comprehensive biochemical identification of off-target sites with independent cell-based measurements of activity at those sites when assessing nuclease activity and specificity.
Date: 
June 28, 2017
Where: 
HSW 1057 at noon

John Gagnon

A maternal-effect selfish genetic element in Caenorhabditis elegans
Ben-David E, Burga A, Kruglyak L.
Science. 2017 Jun 9;356(6342):1051-1055. doi: 10.1126/science.aan0621. Epub 2017 May 11.
Department of Human Genetics, Department of Biological Chemistry, and Howard Hughes Medical Institute, University of California, Los Angeles, CA 90095, USA. [email protected] [email protected] [email protected]
Selfish genetic elements spread in natural populations and have an important role in genome evolution. We discovered a selfish element causing embryonic lethality in crosses between wild strains of the nematode Caenorhabditis elegans The element is made up of sup-35, a maternal-effect toxin that kills developing embryos, and pha-1, its zygotically expressed antidote. pha-1 has long been considered essential for pharynx development on the basis of its mutant phenotype, but this phenotype arises from a loss of suppression of sup-35 toxicity. Inactive copies of the sup-35/pha-1 element show high sequence divergence from active copies, and phylogenetic reconstruction suggests that they represent ancestral stages in the evolution of the element. Our results suggest that other essential genes identified by genetic screens may turn out to be components of selfish elements.
Date: 
June 21, 2017
Where: 
HSW 1057 at noon